Which way does the citric acid cycle turn during hypoxia? The critical role of alpha-ketoglutarate dehydrogenase complex

The citric acid cycle forms a major metabolic hub and as such it is involved in many disease states involving energetic imbalance. In spite of the fact that it is being branded as a "cycle", during hypoxia, when the electron transport chain does not oxidize reducing equivalents, segments of this metabolic pathway remain operational but exhibit opposing directionalities. This serves the purpose of harnessing high-energy phosphates through matrix substrate-level phosphorylation in the absence of oxidative phosphorylation. In this Mini-Review, these segments are appraised, pointing to the critical importance of the alpha-ketoglutarate dehydrogenase complex dictating their directionalities. (c) 2013 Wiley Periodicals, Inc

Cyclosporin A-insensitive permeability transition in brain mitochondria - Inhibition by 2-aminoethoxydiphenyl borate

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca 2 � release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria ex- hibit unique PTP characteristics, including relative re- sistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca 2 � - induced Ca 2 � release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological con- centrations of ATP and Mg 2 � .Ca 2 � release was not me - diated by the mitochondrial Na � /Ca 2 � exchanger or by reversal of the uniporter responsible for energy-depend- ent Ca 2 � uptake. Loss of mitochondrial Ca 2 � was accom - panied by release of cytochrome c and pyridine nucleo- tides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca 2 � -induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A 2 or nitric-oxide synthase but was abol - ished by pretreatment with bongkrekic acid. These find- ings indicate that in the presence of adenine nucleotides and Mg 2 � ,Ca 2 � -induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-amino- ethoxydiphenyl borate

A SUCLA2 mutációk szerepe a mitokondrium foszforilációs rendszerének betegségeiben = The Role of SUCLA2 Mutations in the Disorders of the Mitochondrial Phosphorylation System

Ami a SUCLA2 knock-out egerek használatát illeti, az állatok létrehozása még mindig folyamatban van, annak ellenére, hogy a kifizetés teljes egészében megtörtént. Amint megérkeznek az állatok, munkacsoportunk elsődleges feladata a tervben szereplő, eddig el nem végzett kísérletek végrehajtása lesz. Mindemellett jelentős előrehaladás történt két másik transzgenikus kolónia (DLD +/- és DLST +/-) tekintetében, mint azt a részletes jelentésben is leírtuk. A COS-7 és HEK293 sejteken tervezett siRNS-sel történő SUCLA2 elcsendesítést illetően eddig 4 különböző szekvenciát próbáltunk ki, de nem tapasztaltunk velük elcsendesedést. A citoszolikus/nukleáris ATP mérést illetően egy Japán kutatócsoport 11 plazmidot állított elő, melyekről kimutatták, hogy alkalmasak citoszolikus, nukleáris és mitokondriális ATP mérésére FRET alapú indikátorokként. A plazmidok nemrégiben érkeztek meg, jelenleg a mennyiség növelésén dolgozunk. A (iii) célkitűzést illetően, mely szerint SUCLA2 mutációban szenvedő betegek fibroblasztjain kísérletezünk: ezeket telomeráz gén általi immortalizálás nélkül felhasználtuk. Ahogyan azt az előzetes eredmények alapján is jósoltuk, azt találtuk, hogy SUCLA2 mutációban szenvedő betegekből származó fibroblasztok mitokondriumai gátolt légzési lánc mellett depolarizációt mutatnak bongkreksav hatására, ami az ANT korai megfordulására utal. | Regarding the aim using SUCLA2 knock out mice, this transgenic animal is still on the process of being generated, even though it has been paid in full. As soon as the mice will arrive in our facility, it will be a top priority to perform all remaining proposed experiments. However, significant progress on two other transgenic mouse colonies, DLD+/- and DLST+/-, as mentioned in the detailed progress report. Regarding siRNA against SUCLA2 in COS-7 and HEK293 cells, we have tried 4 different sequences; however, we have not observed any silencing. Regarding measurements of cytosolic/nuclear ATP, 11 plasmids have been generated by a Japanese group, shown to report cytosolic, nuclear, or mitochondrial ATP levels, operating as FRET-based indicators we have just obtained the plasmids, and we are currently expanding them in quantities. Regarding aim (iii), using fibroblasts from patients suffering from SUCLA2 mutations, we used them as such without immortalizing them by inserting a telomerase gene. Our findings showed that -as predicted by the preliminary results- fibroblasts obtained from patients suffering from SUCLA2 mutations exhibited bongkrekic acid-induced depolarizations in respiration-impaired in situ mitochondria, implying premature ANT reversals

Exacerbated responses to oxidative stress by an Na+ load in isolated nerve terminals: the role of ATP depletion and rise of [Ca2+](i)

We have explored the consequences of a [Na 1 ] i load and oxidative stress in isolated nerve terminals. The Na 1 load was achieved by veratridine (5–40 m M ), which allows Na 1 entry via a voltage-operated Na 1 channel, and oxidative stress was induced by hydrogen peroxide (0.1–0.5 m M ). Remarkably, nei- ther the [Na 1 ] i load nor exposure to H 2 O 2 had any major effect on [Ca 2 1 ] i , mitochondrial membrane potential ( D c m), or ATP level. However, the combination of an Na 1 load and oxidative stress caused ATP depletion, a collapse of D c m, and a pro- gressive deregulation of [Ca 2 1 ] i and [Na 1 ] i homeostasis. The decrease in the ATP level was unrelated to an increase in [Ca 2 1 ] i and paralleled the rise in [Na 1 ] i . The loss of D c m was prevented in the absence of Ca 2 1 but unaltered in the presence of cyclosporin A. We conclude that the increased ATP con- sumption by the Na,K–ATPase that results from a modest [Na 1 ] i load places an additional demand on mitochondria met- abolically compromised by an oxidative stress, which are un- able to produce a sufficient amount of ATP to fuel the ATP- driven ion pumps. This results in a deregulation of [Na 1 ] i and [Ca 2 1 ] i , and as a result of the latter, collapse of D c m. The vicious cycle generated in the combined presence of Na 1 load and oxidative stress could be an important factor in the neuro- nal injury produced by ischemia or excitotoxicity, in which the oxidative insult is superimposed on a disturbed Na 1 homeostasis

Mitochondrial consumption of cytosolic ATP: Not so fast

AbstractIn various pathologic circumstances depolarized mitochondria are thought to precipitate cell death by avidly consuming cytosolic ATP. However, for as long as the inner mitochondrial membrane remains intact the reversal potentials of the adenine nucleotide translocase (ANT) and that of F0–F1 ATP synthase are strategically positioned so that they oppose import of cytosolic ATP into the matrix of respiration-impaired mitochondria. This arrangement also seems to protect against a hysteretic consumption of cytosolic ATP accumulating in the mitochondrial matrix, in view of the depolarization caused by inhibition of F0–F1 ATP synthase by the endogenous protein IF1, yielding fast ANT reversal rates

Quantification of mitochondrial DNA from peripheral tissues

Neurometabolic disorders stem from errors in metabolic processes yielding a neurological phenotype. A subset of those disorders encompasses mitochondrial abnormalities partially due to mitochondrial DNA (mtDNA) depletion. mtDNA depletion can be attributed to inheritance, spontaneous mutations or acquired from drug-related toxicities. In the armamentarium of diagnostic procedures, mtDNA quantification is a standard for disease classification. However, alterations in mtDNA obtained from peripheral tissues such as skin fibroblasts and blood cells do not often reflect the severity of the affected organ, in this case, the brain. The purpose of this review is to highlight the pitfalls of quantitating mtDNA from peripheral -and not limited to-tissues for diagnosing patients suffering from a variety of mtDNA depletion syndromes exhibiting neurologic abnormalities. In lieu, a qualitative test of mitochondrial substrate-level phosphorylation -even from peripheral tissues-reflecting the ability of mitochondria to rely on glutaminolysis in the presence of respiratory chain defects is proposed as a novel diagnostic assessment of mitochondrial functionality

Mitochondrial permeability transition pore: Back to the drawing board

Current models theorizing on what the mitochondrial permeability transition (mPT) pore is made of, implicate the c-subunit rings of ATP synthase complex. However, two very recent studies, one on atomistic simulations and in the other disrupting all genes coding for the c subunit disproved those models. As a consequence of this, the structural elements of the pore remain unknown. The purpose of the present short-review is to (i) briefly review the latest findings, (ii) serve as an index for more comprehensive reviews regarding mPT specifics, (iii) reiterate on the potential pitfalls while investigating mPT in conjunction to bioenergetics, and most importantly (iv) suggest to those in search of mPT pore identity, to also look elsewhere. © 2017 Elsevier Ltd

Acute sources of mitochondrial NAD+ during respiratory chain dysfunction

It is a textbook definition that in the absence of oxygen or inhibition of the mitochondrial respiratory chain by pharmacologic or genetic means, hyper-reduction of the matrix pyridine nucleotide pool ensues due to impairment of complex I oxidizing NADH, leading to reductive stress. However, even under these conditions, the ketoglutarate dehydrogenase complex (KGDHC) is known to provide succinyl-CoA to succinyl-CoA ligase, thus supporting mitochondrial substrate-level phosphorylation (mSLP). Mindful that KGDHC is dependent on provision of NAD+, hereby sources of acute NADH oxidation are reviewed, namely i) mitochondrial diaphorases, ii) reversal of mitochondrial malate dehydrogenase, iii) reversal of the mitochondrial isocitrate dehydrogenase as it occurs under acidic conditions, iv) residual complex I activity and v) reverse operation of the malate-aspartate shuttle. The concept of NAD+ import through the inner mitochondrial membrane as well as artificial means of manipulating matrix NAD+/NADH are also discussed. Understanding the above mechanisms providing NAD+ to KGDHC thus supporting mSLP may assist in dampening mitochondrial dysfunction underlying neurological disorders encompassing impairment of the electron transport chain